| BackgroundHepatocellular carcinoma（HCC）is ranked as the third leading cause of cancer-related death worldwide and is the main event leading to death in patients with liver cirrhosis;worryingly,the incidence is increasing.Chronic hepatitis B virus（HBV）infection is the most common cause of HCC in China.Although a multitude of diagnostic and therapeutic modalities have been developed for HCC,the long-term prognosis remains poor.Because,by the time of diagnosis,most HCC cases have developed to late stage and missed the best opportunity to receive optimal therapy.Therefore,early diagnosis and treatment is essential for improving the prognosis and survival rate of patients with HCC.Hepatic histopathological examination is the most reliable method for the diagnosis of HCC,but it is an invasive method and is not suitable for early screening of HCC.A combination of imaging technologies and determination of alpha-fetoprotein（AFP）levels is the most widely used method for detecting HCC.However,low sensitivity and high rates of both false negatives and false positives limit the wide application of serum AFP levels as a marker.The surveillance ability of imaging technologies relies largely on examiner expertise,and size of the tumor,which affects the effective and timely detection of HCC at its initial stage.In addition,post-surgical recurrence,an extremely complex multistep process,is an important cause of poor prognosis in HCC patients after hepatectomy.Several studies have reported that various clinicopathological factors,such as tumor size,tumor number,tumor differentiation,and portal vein invasion,can affect postoperative recurrence and prognosis of HCC patients.HCC patients with the same clinicopathological characteristics often have different prognostic status,which limits its application in the determination of postoperative recurrence and prognosis of HCC.Therefore,there is an urgent need for development of a robust biomarker to improve early diagnosis of HCC and predict the prognosis of HCC and guide effective treatment to improve the prognosis.The occurrence and development of HCC is a multifactorial process.A lot of studies have shown that epigenetic alteration is closely related to the occurrence and metastasis of a variety of tumors.DNA methylation,as one of the most extensively investigated epigenetic DNA modifications,is an important epigenetic regulatory mechanism for gene expression.DNA methylation occurs generally in CpG islands of gene promoters regions,and can trigger aberrant gene expression and inactivation of suppressor genes.Similar to other tumors,carcinogenesis and progression of HCC is driven by complex genetic and epigenetic alterations.A number of studies show that cancer-linked DNA methylation is closely related to initiation,progression,and invasion of HCC.Therefore,abnormal methylation of specific genes can be used as an indicator for early diagnosis and prognosis of tumors.In recent years,the relationship between oxidative stress and progression of HCC has attracted more and more attention.Accumulating evidence has shown that oxidative stress plays a crucial role in the development of HCC.Oxidative stress,an imbalanced state,is caused by excessive reactive oxygen species production and/or a decrease in antioxidant defenses.The carcinogenic potential of oxidative stress can be attributable to the genotoxic effects of reactive oxygen species,which are capable of causing epigenetic alterations in cancer-related genes and leading to HCC.Cyclin D2（CCND2）is a member of the D-type cyclin family.Its main function is to form a complex with cyclin-dependent kinase（CDK）4 and CDK6;this complex induces phosphorylation of retinoblastoma（RB）,thereby prompting cells to enter S phase.In addition to its role in the G1/S transition during the cell cycle,CCND2 may also function to maintain cells in the non-proliferative state and to promote exit from the cell cycle.The alternative function of CCND2 has been proven in several studies.Hypermethylation of the CCND2 promoter is reported to lead to transcriptional silencing of CCND2.This function may be circumvented by down-regulation of CCND2 expression via promoter hypermethylation,which frequently occurs in HCC.It has been reported that transcriptional silencing of CCND2 is closely related to the occurrence and development of breast cancer,prostate cancer,gastric cancer and pancreatic cancer.Based on these observations,we analyzed the methylation status of the CCND2 promoter in patients with HCC and assessed the value of CCND2 promoter methylation as a non-invasive method for diagnosing patients with HBV-associated HCC.We investigated the level of CCND2 methylation in HBV associated HCC and evaluated its prognostic significance in HCC patients undergoing hepatectomy.We investigated the oxidative stress status in HCC patients,and explored the correlation between CCND2 promoter methylation and levels of oxidative stress factors in HBV-related HCC patients.PART Ⅰ Diagnostic Value of Cyclin D2 Gene Methylation in Hepatitis B Virus-Associated Hepatocellular CarcinomaObjectivesIn this study,we aimed to detect the methylation status of the CCND2 promoter in both plasma and PBMCs,and assess the value of CCND2 promoter methylation as a non-invasive method for diagnosing patients with HBV-associated HCC.MethodsA total of 118 patients with HCC,47 with liver cirrhosis（LC）,75 with chronic hepatitis B（CHB）,and 35 healthy controls（HCs）were recruited in the Department of Hepatology,Qilu Hospital of Shandong University,from March 2018 to December 2019.All patients were HBsAg-positive.We measured methylation status and mRNA levels of CCND2 in plasma and PBMCs,using methylation-specific PCR（MSP）and quantitative real-time PCR（RT-qPCR）.Results1.CCND2 mRNA levels were significantly lower in HCC patients than in LC patients（P<0.001）,CHB patients（P<0.001）,and HCs（P<0.001）.However,there were no significant differences in CCND2 mRNA levels between LC patients,CHB patients,and HCs（P>0.05,respectively）.Moreover,HCC patients with advanced disease（TNM stage Ⅲ/Ⅳ）had significantly lower CCND2 mRNA levels than patients with early-stage disease（TNM Ⅰ/Ⅱ）（Z=-2.174,P=0.030）.CCND2 mRNA levels in the HCC group exhibited no significant relationship with age,HBeAg,gender,smoking status,alcohol use,AFP,tumor number,tumor size,vascular invasion,or CTP staging（P>0.05）.2.The frequency of CCND2 promoter methylation was significantly higher in HCC patients（55/118,46.61%in PBMCs;52/118,44.07%in plasma）than in LC patients（8/47,17.02%in PBMCs;5/47,10.64%in plasma;P<0.001）,CHB patients（8/75,10.67%in PBMCs;7/75,9.33%in plasma;P<0.001）,and HCs（4/35,11.43%in PBMCs;2/35,5.71%in plasma;P<0.001）.However,CCND2 methylation frequencies did not differ significantly between LC patients,CHB patients,and HCs（P>0.05,respectively）.3.To determine whether altered promoter methylation could affect CCND2 transcription,we compared CCND2 mRNA levels in subjects with and without promoter methylation.In the HCC group,the level of CCND2 mRNA was significantly lower in methylated subjects than in unmethylated subjects（Z=-2.045,P=0.041）.4.The methylation frequency of the CCND2 promoter in HCC patients was significantly higher in HCC patients with vascular invasion than in those without vascular invasion（P=0.012 in PBMCs;P=0.008 in plasma）.CCND2 promoter hypermethylation was more common in HCC patients with advanced disease（TNMⅢ/Ⅳ）than in those with early-stage disease（TNM Ⅰ/Ⅱ;P=0.004 in PBMCs;P=0.001 in plasma）.In addition,the CCND2 methylation rate increased gradually with TNM stage.5.For discrimination of HCC from LC,CCND2 promoter methylation had a sensitivity of 46.61%in PBMCs and 44.07%in plasma,and a specificity of 82.98%in PBMCs and 89.36%in plasma.For discrimination of HCC from CHB,CCND2 promoter methylation had a sensitivity of 46.61%in PBMCs and 44.07%in plasma,and a specificity of 89.33%in PBMCs and 90.67%in plasma.Next,we explored the diagnostic value of combined measurement of CCND2 methylation and AFP for HCC patients.For discriminating HCC from LC,the AUC of combined measurement of CCND2 promoter methylation and AFP level was significantly higher than that of AFP alone（0.698 vs 0.540 in PBMCs,P<0.001;0.694 vs 0.540 in plasma,P<0.001）.Furthermore,measurement of CCND2 methylation plus AFP had a sensitivity of 82.20%（97/118）in PBMCs and 81.36%（96/118）in plasma;a specificity of 57.45%（27/47）in both PBMCs and plasma;a PPV of 82.91%（97/117）in PBMCs and 82.76%（96/116）in plasma;and a negative predictive value（NPV）of 56.25%（27/48）in PBMCs and 55.10%（27/49）in plasma.Notably,the sensitivity of combined measurement was significantly higher than that of AFP alone（P<0.001）.For discriminating HCC from CHB,the AUC of combined measurement was also significantly higher than that of AFP alone（0.724 vs 0.571 in PBMCs,P<0.001;0.720 vs 0.571 in plasma,P<0.001）.Moreover,combined measurement had higher sensitivity（82.20%vs 57.63%in PBMCs,P<0.001;81.36%vs 57.63%in plasma,P<0.001）and NPV（69.12%vs 48.98%in PBMCs,P=0.010;68.12%vs 48.98%in plasma,P=0.014）than AFP alone.Last,we found that the HCC detection rate in the CCND2-methylated group was significantly higher than that in the CCND2-unmethylated group,regardless of whether the AFP level was≤20 ng/mL or>20 ng/mL（χ2=14.246,P<0.001,AFP≤20 ng/mL;χ2=5.499,P=0.018,AFP>20 ng/mL）.Conclusions1.Methylation of the CCND2 promoter is common in patients with HCC.Combined measurement of CCND2 methylation plus serum AFP levels increased the diagnostic value of AFP for discrimination of HCC from LC and CHB,indicating that combined detection of CCND2 methylation and AFP has potential as a robust and non-invasive biomarker for diagnosis of HCC.2.CCND2 promoter methylation was observed more frequently in HCC patients with advanced TNM stage and vascular invasion,suggesting that this marker might be used to predict the progression of HBV-associated HCCPART Ⅱ Hypermethylation of Cyclin D2 Predicts Poor Prognosis of Hepatitis B Virus-Associated Hepatocellular Carcinoma after HepatectomyObjectivesThis study aimed to investigate the level of CCND2 methylation in HBV associated HCC by using MethyLight and to evaluate its prognostic significance in HCC patients undergoing hepatectomy.MethodsIn all,257 subjects attending the Qilu Hospital of Shandong University between February 2013 and April 2016 were enrolled.The cohort comprised 166 consecutive HCC patients undergoing surgical resection,61 patients with chronic hepatitis B（CHB）,and 30 healthy controls（HCs）.All HCC patients were HBsAg-positive,diagnosed according to the 2010 update of the American Association for the Study of Liver Diseases Practice Guidelines for Management of HCC.All patients were followed up from the date of surgery to March 2019.Early tumor recurrence（ETR）was defined as recurrence within 1 year after curative hepatectomy.CCND2 methylation level was quantitatively measured using MethyLight and CCND2 mRNA level was measured by using quantitative real-time PCR（RT-qPCR）.Survival time between groups was determined by Kaplan-Meier method and compared by log-rank test.Multivariate analyses were performed by Cox proportional hazards models to investigate those prognostic factors that predicted overall survival（OS）,disease-free survival（DFS）and ETR.Results1.The percentage of methylated reference（PMR）values were used to express CCND2 methylation level.The methylation level of CCND2 in HBV-associated HCC patients（median 27.26%,interquartile range 14.41-52.76%）was significantly higher than that in CHB patients（median 5.33%,interquartile range 2.38-10.33%,P<0.001）and HCs（median 4.59%,interquartile range 2.83-6.60%,P<0.001）.There were no significant difference in methylation level between CHB patients and HCs（P>0.05）.Furthermore,we found a negative correlation between CCND2 methylation level and its mRNA expression level in patients with HCC（r=-0.46,P<0.001）.2.To evaluate the clinicopathological significance of CCND2 methylation,we explored the association between CCND2 methylation level and the clinicopathological features of HCC patients.CCND2 methylation level was significantly higher in HCC patients with portal vein invasion（median 39.78%,interquartile range 22.93-70.96%）than in those without vascular invasion（median 26.42%,interquartile range 12.54-43.83%,P<0.01）,in patients with early tumor recurrence（ETR）（median 54.71%,interquartile range 24.15-72.70%）than in those without ETR（median 22.69%,interquartile range 12.24-39.50%,P<0.001）,in patients with TNM Ⅲ/Ⅳ stage（median 39.78%,interquartile range 22.93-70.96%）than in those with TNM Ⅰ/Ⅱ stage（median 23.00%,interquartile range 11.99-40.34%,P<0.001）,and in patients with tumor size≥ 5 cm（median 30.57%,interquartile range 19.35-55.29%）than in those with tumor size<5 cm（median 21.40%,interquartile range 12.12-43.83%,P=0.013）.3.We constructed Kaplan-Meier curves to examine correlations between CCND2 methylation and outcomes of HCC patients.We selected the median PMR value of 27.26%as the cutoff point in distinguishing high and low CCND2 methylation level in HCC patients.The time for OS in higher methylation level group was significantly shorter than in lower group（P=0.005）.In addition,patients with higher CCND2 methylation level had poorer DFS（P<0.001）than those with lower CCND2 methylation level.4.Next.we used univariate and multivariate analyses to identify factors prognostic for OS and DFS.Univariate analyses identified tumor number,tumor size,portal vein invasion,TNM stage,and CCND2 methylation level as being associated with OS and DFS.All five variables were entered into multivariate analyses using Cox proportional hazards models.The results identified TNM stage（HR,3.520;95%CI,1.102-11.240;P=0.034）,and high CCND2 methylation level（HR,1.740;95%CI,1.082-2.799;P=0.022）as independent risk factors for OS;only CCND2 methylation level（HR,2.184;95%CI,1.472-3.240;P<0.001）as independent indicators of DFS.5.Finally,we examined the effects of CCND2 methylation on ETR of HCC.Multivariate analyses identified higher CCND2 methylation level（OR,2.467;95%CI,1.143-5.324;P=0.021）as an independent predictor for ETR.Conclusions1.Hypermethylation of CCND2 was associated with HCC progression and ETR,and served as an independent predictor for OS,DFS and ETR of HCC,indicating that detection of CCND2 methylation may be a non-invasive approach to predicting poor prognosis and ETR in patients with HBV-associated HCC undergoing hepatectomy.2.Hypermethylation of CCND2 may also have clinical value for risk stratification and monitoring of disease progression.PART Ⅲ Relationship between CCND2 Promoter Methylation and Oxidative Stress in PBMCs of Patients with Hepatitis B Virus-Associated Hepatocellular CarcinomaObjectivesThis study aimed to investigate the oxidative stress status in HCC patients,and to explore the correlation between CCND2 promoter methylation and levels of oxidative stress factors in HBV-related HCC patients.MethodsWe enrolled in 166 patients with HBV-related HCC from the Department of Hepatology,Qilu Hospital of Shandong University,between June 2016 to August 2019.In addition,we recruited 40 liver cirrhosis,66 chronic hepatitis B and 30 healthy controls.The HBV-related HCC patients fulfilled the 2010 update of the American Association for the Study of Liver Diseases Practice Guidelines for Management of hepatocellular carcinoma.We detected the methylation status of CCND2 in PBMCs from the hepatitis B virus-related HCC（HBV-related HCC）patients and the healthy controls（HCs）by methylation-specific polymerase chain reaction（MSP）.We also determined the oxidative stress parameter levels in plasma of the patients with HBV-related HCC and HCs by enzyme-linked immunosorbent assay（ELISA）.The differences between continuous variables were analyzed by Mann-Whitney U-test.Categorical values were presented by relative frequencies.The Chi-square test was applied to categorical data.The correlation between difference variables were analyzed by the Spearman correlation.Statistical analysis was performed using SPSS version 19.0 software（SPSS,Chicago,IL,USA）and GraphPad Prism 6.0（San Diego,CA,USA）.Thirty-six patients with HBV-related HCC and 11 HCs were selected for detection of metabolites in plasma by ultrahigh performance liquid chromatography-mass spectrometry（UHPLC-MS）.Data obtained from metabolomics were imported into the SIMCA software（V16.0.2,Umea,Sweden）for principal component analysis（PCA）,orthogonal partial least squares discrimination analysis（OPLS-DA）.Metabolites with P<0.5,variable importance for the projection（VIP）>1,fold change>2 or |
